Inositolphospholipids and analogues: phosphatidylinositol products and processes

ABSTRACT

Embodiments of the invention relate to natural and synthetic inositolphospholipid (IPL) materials, their preparation and applications. They provide compositions of the parent IPL comprising phosphatidylinositol (PI), PI-phosphates (phosphoinositides) and derivatives and analogues, and a process for their production starting from natural IPL. The embodiments further provide functional derivatives of PI for biomedical applications including a platform for drug design and delivery to therapeutic targets in the phosphoinositide mediated cellular signaling and allied cascades. The embodiments pertain to IPL having absolute stereo-structure. The embodiments further pertain to unique IPL and PI product compositions for defined applications, particularly pharmaceutical compositions for prophylaxis and treatment of diseases related to aberrant cellular and nuclear signaling mediated by PI and PI derived phosphates, and associated phosphoinositide specific enzymes including PI-PLC and PI 3-kinase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. provisionalapplication Ser. No. 60/752,542, entitled “Inositolphospholipids AndAnalogues: Phosphatidylinositol Products And Processes,” filed Dec. 21,2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The present invention was partially made with funds provided by theDepartment of Health and Human Services under NIH Grant No. GM59550.Accordingly, the United States Government owns certain rights in thisinvention.

FIELD OF THE INVENTION

Embodiments of the invention pertain to natural and syntheticinositolphospholipids (IPL) materials, their preparation andapplications. It particularly pertains to the parent IPL comprisingphosphatidylinositol (PI), PI-phosphates (phosphoinositides) andderivatives and analogues, and a process for their production. Itfurther pertains to the molecular design of functional derivatives of PIfor biomedical applications including a platform for drug design anddelivery to therapeutic targets in the phosphoinositide mediatedcellular signaling and allied cascades. The embodiments of the inventionfurther pertain to IPL product compositions for defined applications,particularly pharmaceutical compositions for prophylaxis and treatmentof diseases related to aberrant signal via the IPL, particularly thephosphoinositides (phosphatidylinositols and derived phosphates)mediated cellular and nuclear signaling. In all embodiments, it pertainsto natural and synthetic IPL comprising PI and its derivatives andanalogues having absolute stereo-structure.

BACKGROUND OF THE INVENTION

The IPL are conjugates of an inositol moiety that is regio- andstereo-specifically linked by a phosphodiester bridge to a lipid moiety.Natural IPL generally have structures based on 1D-1-myo-inositol;however, structures based on uncommon inositol isomers, for examplescyllo-inositol, are known. Two structural parents in the myo-inositolseries are the glycerolipid-based PI wherein the lipid residue is a1,2-diacylglyceryl-moiety, and, the sphingolipid-basedceramide-phosphoinositols (Cer-PI) wherein the lipid residue is anN-fattyacyl-sphingosine moiety. Cellular PI belong to the1D-1-myo-inositol series and have the1D-1-(1-fattyacyl¹-2-fattyacyl²-sn-glycero-3-phospho)-myo-inositolabsolute stereochemical structure shown. The cellular Cer-PI commonlyhave the 1D-1-myo-inositol stereochemical structure conjugated toceramide moiety with a D-erythro-sphingosine stereostructure.Representative structural parent IPL are shown in FIG. 1. These absolutestereo-structures are susceptible to change during physico-chemicalprocessing.

PI, their radyl analogues with alkylether in place of fattyacyl, Cer-PI,and, their derivatives carrying fattyacyl, glycosyl, and phosphategroups covalently linked to the inositol residue, occur as minorcomponents of biological cells. As the name signifies, synthetic IPL,their structural and stereochemical analogues, and their respectivederivatives and congeners, are prepared by synthesis.

The IPL are biological and biocompatible amphiphilic materials withdiverse roles and uses. The cellular IPL have critical physicochemical,biochemical and physiological functional roles; in particular, PI andderived mono-, di-, and tris-phosphates, the so-calledphosphoinositides, are transducers in vital intracellular and nuclearsignaling and related processes. The natural, as well as the syntheticproducts are useful, broadly, as biochemical reagents in studies on thestructure and function of cell membranes and mechanisms of intracellularsignaling, as reference compounds for analysis of cellular IPL, assubstrates in assays and diagnostics kits for enzymes involved insignaling via the IPL, as lead compounds for the design and developmentof novel drugs for the treatment of disorders caused by aberrantsignaling including diabetes and some cancers, as nutraceuticals anddrugs for central nervous system disorders and cardiac arterialdiseases, for bio-delivery of specific pharmacodynamic fattyacylscovalently incorporated in the phosphoinositide structure, as the lipidcomponent in liposomal delivery vehicles for cytotoxic drugs, bioactivepeptides, proteins and polynucleotides, and in cosmetics formulations.

The embodiments of the present invention particularly provide novel IPLmaterials and compositions, more particularly, PI and structural andstereochemical analogues, and their respective derivatives including butnot limited to phosphate derivatives. The invention embodimentsadditionally provide methods for isolation and purification of IPL,particularly PI, from natural lipid sources, and, further provide anovel process approach for low cost preparation and large-scaleproduction comprising synthesis using natural IPL as starting materials.The present methods of isolation, purification and synthesis uniquelyare designed and validated to ensure that the IPL products of theinvention retain the core structure and absolute stereochemistry of thenatural IPL, in both the myo-inositol and the lipid residues; for PI,this is the1D-1-(1-fattyacyl¹-2-fattyacyl²-sn-glycero-3-phospho)-myo-inositolabsolute stereochemical structure.

DESCRIPTION OF RELATED ART The Inositolphospholipids: Structures,Biological Roles, and Utility

IPL constitute an important group of biological small molecules, whichincludes the structural parents PI and Cer-PI (Carter et al, 1965).Cellular PI belong to the 1D-1-myo-inositol series and have the1D-1-(1-fattyacyl¹-2-fattyacyl²-sn-glycero-3-phospho)-myo-inositolabsolute stereochemical structure. In the radyl analogues (Radyl-PI),the 1-fattyacyl residue is replaced by O-alkyl, and in Cer-PI (andphytoceramide-PI), the 1,2-diacylglycero residue is replaced by aceramide moiety. The corresponding lyso-series of IPL, Lyso-PI, lack the2-fattyacyl and thus have a free hydroxyl group at the glycero-2position. The sphingosyl-phosphoinositols (Sph-PI) lack the amidefattyacyl and have a free 2-amino group in the long chain lipid base,and the fattyacyl may be derivatized further with OH and C═C functionalgroups. Representative structures of the cellular series are shown inFIG. 1.

As noted above, in cellular PI the inositol moiety is in the1D-1-myo-inositol and the glycerol moiety is in the sn-glycero-3-phosphoconfiguration. The glycerol residue is esterified with mixtures ofsaturated long carbon chain fattyacyls, and unsaturated andpolyunsaturated fattyacyls collectively referred to as (poly)unsaturatedfattyacyls. Thus, PI in natural phospholipids are complex mixtures ofmolecular species differing in their fattyacyl composition anddistribution between glycero-1 and -2 positions. In the naturallyoccurring Cer-PI (and phytoceramide-PI) series, the sphingosine/ceramideresidue has the D-erythro stereochemical configuration. Cer-PI showvariable chain length and degree of unsaturation in the two alkyl groupsof the ceramide residue, and may carry additional OH, C═C and/or relatedfunctional groups. Synthetic PI analogues are based on thesn-glycero-3-phospho as well as the sn-glycero-1-phospho configurations,and the synthetic analogues of Cer-PI are based on the D-erythro,L-erythro, D-threo, or L-threo stereochemical configurations in thesphingosine/ceramide residues. Diastereomers are formed by allcombinations of the 1D-1- and 1L-1-myo-inositols with the aforementionedglycerol or sphingosine/ceramide configurations. This invention pertainsto all such stereochemical combinations.

In eukaryotic cells, PI are quantitatively minor but vital components ofmembrane lipids with critical structural and metabolic roles. In thestructural role, PI function akin to the more abundantphosphatidylcholines and phosphatidylethanolamines in membranes (Small,1986) but this has not been studied in detail. In its metabolic roles,PI is the parent participant in the vital PI cycle, which is responsivedirectly to various extra cellular stimuli acting on the cell (Hokin,1985). Agonist stimulated metabolism is mediated by combinations of manyregulatory protein and enzyme families including the PI transferprotein, PI synthase, and phospholipases, kinases andphosphate-phosphatases specific for the PI group implicated inintracellular signaling. Mono-, bis-, and tris-phosphates of PI areformed and their cellular concentrations are regulated by the actions ofkinase and phosphate-phosphatase groups of enzyme families, which arespecific for the 3-, 4-, or 5-positions. The action of PI specificphospholipase C (Rhee et al, 1989) on PI-4,5-bisphosphate generates theintracellular second messengers inositol-1,4,5-trisphosphate anddiacylglycerol which respectively mediate release of intracellular Caions (Berridge, 1984, 1987, 1993) and activation of protein kinase C(Nishizuka, 1986) respectively. The 3-phosphate series (Whitman et al,1988) act as messengers in mitogenic and related signals more directly(Toker et al, 1994; Duckworth and Cantley, 1996). Action of cytosolicphospholipase A₂ liberates arachidonic acid from the sn-glycero-2-O-acylmoiety (Lapetina et al, 1981) which is utilized in the arachidonicacid-eicosanoid messenger cascades. Thus, PI is a direct and indirectreservoir of additional signaling molecules, which mediate and controlvital cellular functions (Bell et al, 1996). PI moiety is the lipidcomponent in glycosyl-PIs which function as membrane anchors ofimportant cellular proteins (Englund, 1993), and as transducers in theinsulin messenger cascade (Saltiel et al, 1986). The radyl and sphingoanalogues of PI and glycosyl-PIs have similar and additional roles(Ferguson and Williams, 1988). Synthetic IPL and analogues are used asresearch reagents in multifarious signaling and related biomedicalfields. PI as the amphiphiles in liposomal drug delivery vehiclesprevents recognition of vesicle surface by the phagocytic cells of thereticuloendothelial system, the circulating mononuclear phagocytic cellsand located in liver and spleen, and enhances blood circulation time ofthe drug formulation (Lee et al, 1992).

Plant PI has a dramatic toxic effect on numerous tumor cells lines butnot on normal cells (Jett et al, 1985). Difference between PI types havebeen attributed to the fattyacyl composition, particularly at thesn-glycero-2-O position (Jett et al, 1985). Plant PI, specifically SoyPI, also was reported to have antiviral activity and recommended as aprophylactic for HIV; the identity of the fattyacyl in thesn-glycero-2-O-position appears critical for activity (Jett-Tilton,1991). Plant PI has beneficial therapeutic effects for central nervoussystem disorders including depressions and pharmacologically inducedmemory alterations (Ferrari, 1993, U.S. Pat. No. 5,214,180).

More recently, attention has been on developments based on inhibitorsand modulators of the phospholipases, kinases, and phosphatephosphatases involved in signal transduction. PI analogues modified inthe inositol-2-position, for instance the 2-fluorodeoxy-scyllo-inositoltypes, were found to be effective inhibitors of phospholipase C andpotent anti-inflammatory and analgesic agents (Yang et al, 1985).2-Modified phosphoinositides in general have analogous potential as leadcompounds for the development of therapeutics relying on the inhibitionof PI-specific phospholipase C (PI-PLC) (Aneja and Aneja, 1999). Otherinhibitors of PI-PLC are based on the thiophosphate and phosphonateanalogues. The water soluble D-myo-inositol4-(hexadecyloxy)-3(S)methoxy-butane-phosphonate, a phosphonate analogueof PI, is reported to inhibit epithelial cell proliferation (Leung etal, 1998a). PI analogues modified at the inositol-3-position inhibit thegrowth of mammalian cells, and have potential for treating neoplasticconditions and other proliferative disorders (Kozikowski et al, 1993,U.S. Pat. No. 5,227,508). Inhibitors of ceramide-phospho-inositolsynthase are potent antifungal agents (Mandala et al, 1998).

Early studies on the effects of PI on serum lipids and lipoproteins inrelation to cardiac arterial disease (CAD), especially mobilization ofcholesterol to the blood stream and development and resorption ofatherosclerosis, gave conflicting results (Sachs et al, 1960, andreferences therein). Very recently, it was reported that administrationof PI stimulates reverse cholesterol transport by increasing the flux ofcholesterol into high density lipoprotein-cholesterol (HDL-C), raisesHDL-C levels in humans, and suggesting that PI administration has atherapeutic benefit in CAD (Sparks, 2004, U.S. Pat. No. 6,828,306;Burgess et al, 2005).

Significant quantities of IPL, particularly PI, derivatives andanalogues are required for aforementioned application areas, includingbut not limited to use as drug delivery vehicles, nutraceuticals,therapeutics, and in the drug cum drug delivery vehicles based on PIthat are an integral part of the present invention. All known andupcoming future applications mandate that the IPL and PI products haveunequivocally established stereo-structures. In addition, large-scaleapplications including drug delivery vehicles, nutraceuticals andtherapeutics mandate a low production cost. The prior art has notprovided natural source PI, particularly plant source PI withunequivocally validated absolute stereo-structure, and at low costproduction.

The Preparation of Natural and Synthetic Phosphatidylinositols—Prior ArtRelevant to Soy PI

Prior art methods for preparing Natural PI from plant sources areoutlined below. Recent and prior art on synthesis has been reviewedextensively by the Inventor (Aneja, 2004, U.S. Pat. No. 6,737,536), andis incorporated herein by reference.

Natural PI-enriched fractions have been isolated from soy lecithins bysolvent extraction and liquid-liquid partition but the productsinvariably contain phosphatidylethanolamines (PE) and phosphatidic acid(PA) (Shimizu et al, 1992, U.S. Pat. No. 5,100,787).

A PI-enriched fraction, named lipositol, prepared by multipleprecipitations using cold CHCl₃ and CH₃OH (MeOH), had a significantNitrogen content, ascribable to PE (Woolley, 1943); it was confirmedlater that the major contaminant in such preparations is PE (Colaciccoand Rapport, 1967).

PI-enriched lecithin fractions were prepared by countercurrentdistribution but had significant PE, phosphatidylcholine (PC), andallied impurities (Carter and Kisic, 1969).

Relatively pure soy PI was obtained by a composite process comprisingsolvent extraction, ion exchange on a Chelex 100 resin, columnchromatography on silica, and “crystallization” (Colacicco and Rapport,1967). These, and allied methods using chromatography ondialkylaminoalkyl-ion exchange media diethylaminoethyl cellulose andrelated matrices, are tedious, costly and not suitable for large-scaleproduction.

A process for recovering PI from grain powders initially produces aPI-protein complex, from which PI is isolated by extraction; it issurprising that the extracting solvent is said to be methanol (Gilmanovet al, 1990, U.S. Pat. No. 4,977,091).

Another process for the preparation of highly pure PI (Ferrari et al,1993, U.S. Pat. No. 5,214,180), initially converts the PE in lecithin toPC by treatment with base and CH₃I, and removes PC by solventextraction. The residue is treated with reagents selected from the groupconsisting of dimethyltertbutylsilylchloride,thexyldimethylsilylchloride, trimethylsilylchloride and allyl bromide,to convert PI into alkylsilyl or allyl derivatives. Finally, thealkylsilyl or allyl groups are removed under conditions such as not tomodify the phosphatidyl group, but proof about changes, if any, to theabsolute stereo-structure of the recovered PI was not provided.

In another process, the starting material is a lecithin, which containsPI as the only IPL material. A PC-free lecithin fraction is firstobtained from the starting material by multiple precipitations usingCHCl₃ and MeOH. This fraction is lipolysed with phospholipase D (PLD) toconvert the residual PE into PA, followed by reaction with a phosphataseto convert the PA into diacylglycerol; the resulting mixture of PI anddiacylglycerol is separated by solvent extraction and precipitation(Shimizu et al, 1992, U.S. Pat. No. 5,100,787). The diastereomericpurity of the PI product is uncertain because of other reports showingthat PI is readily attacked by PLD causing hydrolysis andtransphosphatidylation; the transphosphatidylation reaction occurs withPI (as an alcohol) and added alcohols. For instance, it has beenreported that reaction between PI and PLD, produces some PA byhydrolysis, but the main products are bis(phosphatidyl)inositols, formedby inter-molecular transphosphatidylation between two PI substratemolecules (Clarke et al, 1981). Accordingly, concomitant intra-moleculartransphosphatidylation of PI is expected leading to products containingunchanged PI and PI-isomers and diastereomers.

Overall, prior art processes do not disclose that the product PI retainsthe absolute stereo-structure of natural PI. To date, none of the priorart methods has been adapted for the production of natural PI.

SUMMARY OF THE INVENTION

The inositolphospholipids (IPL) are conjugates of an inositol moiety,usually a myo-inositol moiety, linked regio- and stereo-specifically bya phosphodiester bridge to a lipid moiety, commonly a1,2-difattyacyl-glycerol or ceramide. The cellular IPL have criticalphysicochemical, biochemical and physiological functional roles, forexample as transducers in vital intracellular and nuclear signaling.Natural and synthetic IPL have diverse uses in biomedicine, nutritionand food, as reagents for signaling research, enzyme assays, drugdiscovery, drug delivery, nutraceuticals, and therapeutics.

An embodiment of the invention pertains to natural and synthetic IPLcomprising PI and its derivatives and analogues for which unequivocalproof of absolute stereo-structure is provided herein. In oneembodiment, this invention relates to natural IPL, their preparation andapplications. The invention provides natural IPL materials that aresubstantially free of non-IPL materials, and have unequivocallyestablished core IPL stereo-structure. The invention particularlyprovides IPL products that are substantially free of PE and PA. Thenatural IPL products include compositions enriched in the structuralparent PI for which the core absolute stereo-chemical structure is1D-1-(1-fattyacyl¹-2-fattyacyl²-sn-glycero-3-phospho)-myo-inositol.

Concomitantly, an embodiment of the invention provides a low costprocess for the isolation and purification of the said IPL from naturallipid sources, especially plant seed oil phosphatides and commerciallecithins, and, in a particular aspect, soy lecithins. The processembodies parameters, which demonstrably preserve the corestereo-structure of the natural IPL, including the core absolutestereo-chemical structure1D-1-(1-fattyacyl¹-2-fattyacyl²-sn-glycero-3-phospho)-myo-inositol ofPI.

Another embodiment further provides unique IPL product compositions foruse broadly in biomedical applications; the unique compositions are usedas such or as blends with selected materials.

Another embodiment further provides unique IPL product compositions usedas starting materials for preparation of natural and synthetic IPLs andrationally designed analogues including PI and derivatives for definedapplications.

The embodiments of the invention provide an advantageous low costprocess for production of natural IPL, particularly soy PI, comprisingisolation from natural lipid sources, and for the conversion of naturalIPL into semi-synthetic derivatives and rationally designed novelanalogues. It further provides novel natural and synthetic IPL materialsand compositions, made directly by the present process, with or withoutadmixing with other lipids and materials.

The embodiments of the invention also provide specific IPL productsincluding soy PI, and its derivatives and analogues carrying additionalfunctional groups or substituents with negative, positive or neutral netcharge, including phosphate, thiophosphate, sulphate, carboxylate,ω-aminoalkyl, alkyl, and polyethyleneglycol, and their respectiveisosteric molecular and functional group analogues, exemplified by butnot limited to the structures shown in FIG. 2, and their cell-permeablederivatives and complexes.

The embodiments of the invention further provides novel syntheticPI-based structural classes carrying substituents at theinositol-6-O-position, with or without additional substituent(s) ormodification(s) at one or more remaining four inositol hydroxyls (FIG.2). These PI-based structural classes and libraries are designed asinhibitors of therapeutic targets in the PI and derived PI-phosphate(phosphoinositide) mediated enzyme and effector protein signalingcascades, and as modulators of the biological activity of PI- andphosphoinositide-dependent lipoproteins.

The embodiments of the invention also provide a novel approachintegrating drug design and drug delivery, specifically to therapeutictargets in the phosphoinositide and allied metabolic and signalingcascades. This approach is based on a novel molecular design comprisingtherapeutically active IPL delivery vehicles that are conjugated, via acontrolled stability and adjustable chain length linker, to another drug(FIG. 3). The conjugates retain the intrinsic propensity of the IPL foraccumulation in vivo in specific lipid-protein complexes and forclustering in special cell membrane domains and thus provide templatesfor binding specific effector proteins and signaling enzymes. The IPLresidue has inherent drug bioactivity towards defined therapeutictargets that include the aforementioned effector proteins and enzymes inthe phosphoinositide-dependent metabolic and intracellular signalingpathways. The other drug is bioactive towards therapeutic targets in thephosphoinositides cascade or downstream from thephosphoinositide-dependent metabolic and intracellular signalingpathways; the latter include protein kinases, phosphatases and alliedsignaling proteins. The physical dimensions, flexibility, and propensityof the linker towards chemical or biochemical rupture ensure that theother drug is either held or released in spatial proximity to thetherapeutic targets. This invention specifically provides the said IPLdelivery vehicles comprising PI-derivatives conjugated to controlledstability and adjustable chain length linkers, and to second drug.

The embodiments of the invention also provide selectively O-protected PIderivatives that are key precursors and intermediates in synthesis andare utilized herein for the preparation of natural IPL, exemplified bysoy PI and Soy PI-4,5-bisphosphate (FIGS. 4, 5 and 6), and theaforementioned rationally designed IPL derivatives and analogues.

Thus, it will be seen that the embodiments of the invention provide theaforementioned novel IPL compositions, particularly PI, derivatives andanalogues, O-protected PI derivatives, all with unequivocalstereo-structures.

The embodiments of the invention provide a scalable low cost processapproach for the preparation and production of the aforementioned novelIPL, particularly PI, derivatives and analogues, O-protected PIderivatives, all with unequivocally established stereo-structures.

The embodiments of the further pertain to novel unique IPL productcompositions for defined applications, particularly pharmaceuticalcompositions for prophylaxis and treatment of diseases related toaberrant signal via the IPL, particularly the phosphoinositides(phosphatidylinositols and derived phosphates) mediated cellular andnuclear signaling.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows representative structures of the cellular series parentIPL;

FIG. 2. shows representative structures of PI derivatives carryingadditional functional groups;

FIG. 3. shows PI-based vehicles and drugs for specific delivery totherapeutic targets in the phosphoinositide and allied metabolic andsignaling cascades;

FIG. 4. shows conversion of Soy PI into PI-4,5-bisphosphate;

FIG. 5. shows purification of soy PI via protection toDi-O-Cyclohexylidene-PI isomers, and deprotection to PI;

FIG. 6. shows purification via protection to Penta-O-protected-PI anddeprotection to PI; and

FIG. 7. shows acid catalyzed phosphatidyl migration and isomerization.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION NaturalSource Lipid Starting Materials

Starting materials for the invention are natural source lipids thatcontain IPL. These materials are exemplified by phosphatide gums fromplant seed oils and the derived commercial lecithins. Plant seedphosphatides are by-products in the production of refined edible oils,and are obtained initially as hydrated gums in the steam-refining step;these are used directly or after dehydration. The dehydrated phosphatidegums are available as commercial plant seed lecithins. Soybean lecithinsoften are referred to as soy lecithins or simply as lecithins.

Lecithins are available from several commercial sources, and are thepreferred starting materials. Commercial lecithins are complex mixturesof natural lipids, and are produced as fluid (liquid), and powder orgranulated forms. Fluid lecithins contain triglyceride (TG) as asubstantial component, which is removed by selective extraction withacetone or by supercritical fluids to produce de-oiled lecithins. Thefollowing composition data for commercial lecithins are typical and aretaken from the brochure “Learn about Lecithins” published by AmericanLecithin Company, Oxford, Conn., U.S.A.

-   -   Fluid lecithins: Triglycerides (TG, 37%); Phosphatidylcholines        (PC, 16%); Phosphatidylethanolamines (PE, 13%);        Phosphatidylinositol (PI, 10%); Phosphatidic Acid (PA, 5%);        Minor phospholipids (unidentified, 2%); Glycolipids (11%);        Complexed Sugars (5%); Water (1%); Soybean Meal Fines (0.1%).    -   De-Oiled Lecithins: TG, 3%; PC, 24%; PE, 20%; PI, 14%; PA, 7%;        Minor phospholipids (unidentified, 3%); Glycolipids (15%);        Complexed Sugars (8%); Water (1%); Soybean Meal Fines (0.2%).

The aforementioned glycolipids and complexed sugars includesteryl-glycosides (SG), steryl-glycoside fattyacyl esters (SGE) (Anejaet al, 1974), and, significant amounts of phytoglycolipids (PGL),ceramide-phosphate-polysaccharides (CPPS), and highly polar glycolipidsthat lack phosphate, and are provisionally identified asglyco-phytosphingolipids (also known as phytocerebrosides) (GSL). PGLare highly glycosylated IPL that have phytosphingosine-based ceramide asthe long chain lipid residue (rather than diacylglycerol residue presentin PI). The structure of soy PGL presumably is closely related to thatproposed for corn PGL wherein the glycosyl residues compriseglucosamine/N-acetylglucosamine, glucronic acid, mannose, and othersugars, and analogues that lack glucosamine/N-acetylglucosamine (Carteret al, 1969). The minor lipids include N-acyl-PE (Aneja et al, 1969),free fatty acids (FFA), and the IPL Lyso-PI.

Other suitable starting materials include lecithin fractions thatcontain a smaller than typical percentage of PC, PE, PA, and/or PGL andconsequently a higher content of PI (Aneja et al, 1971; Aneja, 1972,U.S. Pat. No. 3,704,254; Aneja and Chadha, 1976, U.S. Pat. No. RE28,903; Ferrari et al, 1993, U.S. Pat. No. 5,214,180). Natural lipidsand fractions containing from about 10 to 50% IPL and PI are preferredstarting materials.

A natural soy lecithin has been mentioned which apparently contains PIas the only IPL component (Shimizu et al, 1992, U.S. Pat. No.5,100,787); however, it is a common experience that such compositionsare based on inadequate analyses for other IPL which are knowncomponents of plant lecithins (Carter et al, 1969). Such lecithincompositions, are desirable starting materials for PI and PI-enrichedproducts.

The Process Approach

The process has a modular design, and comprises selective fractionationof the starting natural lipid mixture by solvent extraction, selectiveprecipitation, and liquid-liquid partition, preferably in conjunctionwith and aided by solvent modifiers and solute solubility modifiers;optionally, an adsorption step is included. Preferably, the products areisolated in sodium salt form. The process parameters are adjustable toobtain desired product compositions and purity. As an additional option,the PI in the IPL material is converted into a derivative, and thederivative is isolated and purified, and finally reconverted intopurified PI product.

Process Design for Lecithins as Starting Materials

This embodiment aspect of the invention is illustrated using soybeanphosphatides (commercial lecithin) as the natural lipid source.

It is well known in the art that the PC component in lecithins issoluble in short chain alcohols, particularly ethanol and aqueousethanol, while PE is sparingly soluble and PI and other IPL arepractically insoluble. Thus, extraction of lecithins with alcoholproduces alcohol-insoluble lecithin fractions which are richer in PI andIPL content than lecithin, but the prior art products invariably containsignificant amounts of PE (Carter and Kisic, 1969, and referencestherein). We now have discovered that use of certain solubilitymodifiers can make PE soluble in short chain alcohols without effect onsolubility of PI, and that extraction of lecithin using alcohols inconjunction with and aided by solubility modifiers simultaneouslydissolve out PE and PC. Additionally, we have discovered that the samesolubility modifiers increase the solubility of PA. This discovery hasprovided a critical basis for the design and development of presentprocess for isolation and purification of IPL and PI from lecithins.Fatty acid anhydrides and equivalent chemical or enzymatic N-acylatingreagents are employed as solubility modifiers, with butanoic anhydrideand acetic anhydride as the preferred reagents; the latter is the mostpreferred, inter alia because of low cost, desirable solubility andrelated physical properties, and the fact that its only by-product isacetic acid.

The process includes three main (Fractionation) and two ancillary(Characterization and Finishing) modules. Further, the process includesan optional module requiring temporary reversible chemical modificationof PI in the starting lipid materials.

These modules are identified below and, thereafter, described in detail.

-   -   Module 1. Selective Solvent Extraction Aided by Modifiers    -   Module 2. Selective Solvent Partition    -   Module 3. Selective Precipitation, Adsorption on Silica,        Crystallization    -   Module 4. Characterization    -   Module 5. Finishing    -   Module 6. Purification of IPL via Temporary Reversible Chemical        Modification        Module 1. Selective Solvent Extraction Aided by Modifiers

The starting phosphatides or Soy lecithin, or a solution thereof, istreated with acetic anhydride, the reaction mixture is poured slowlyinto stirred ethanol, and the precipitated alcohol-insoluble material isseparated from the alcohol-soluble extract solution. The foregoingextraction and separation sequence is next carried out using theprecipitate, or a solution thereof, and is repeated with each successiveprecipitate, usually from 2-6 times; acetic anhydride is employed in oneto three extractions. The process is complete when analysis shows thatthe precipitate is substantially free of components that are less polarthan PI, as judged by thin layer chromatography (TLC) (larger R_(f) thanPI on Silicagel G plates, developed with CHCl₃—CH₃OH—NH₄OH mixtures,typically 60:40:10, v/v/v). The final precipitate is designated as the‘Total-IPL’ product.

The actual yield of Total-IPL ranges from about 50-100% of thetheoretical yield calculated for a virtually complete removal of TG,FFA, SG, SGE, PC, and PE, and a partial removal of PA, and some IPL,from the starting soy lecithin composition.

The exact composition of Total-IPL product depends on the composition ofthe starting material and the conditions for extraction. Typically,Total-IPL contains 20-65% PI, together with PGL, GSL, and minor amountsof PA, and, it is substantially free of TG, FFA, SG, SGE, PC, and PE.This composition specification distinguishes Total-IPL from thealcohol-insoluble fractions of lecithins described in the prior art.

Total-IPL precipitate is processed further in the succeeding modules(Modules 2-5), or utilized in the optional Module 6.

Affect of Acetic Anhydride: Treatment of lecithins with acetic anhydrideconverts any component structures with free amino functional groups intothe corresponding N-acetyl derivatives. Thus, PE is converted intoN-acetyl-PE and TLC (Aneja et al, 1971) detects this change easily.Acetic anhydride-treated lecithins, called ‘acetylated lecithins’, aremarketed in the food additives industry and are considered GRAS. RelatedN-fattyacyl analogues occur, as minor components in plant seed lecithinsand the N-acyl-PEs of soy lecithin have been characterized (Aneja et al,1969).

Under certain conditions in the presence of base or strong acidcatalysts, treatment with acetic anhydride also can acetylate free OHand other functional groups. In the present process module, treatmentwith acetic anhydride is carried out under conditions which exclusivelyacylate free amino groups and preclude esterification of free alcohol OHgroups in PI and other IPL. Reaction is carried out at relatively lowtemperatures (18-40° C.) for a short time (30-90 min), and monitored byTLC for N-acetyl-PE/PE ratio. These preferred conditions wereestablished in model studies; specifically, it was shown that the PIobtained from acetylated lecithins by chromatography, is identical withnatural PI based on rigorous comparison of TLC, MS, NMR and opticalrotation data.

In the present process, neat fluid lecithins are treated with aceticanhydride without or with a partial vacuum, to distill out theby-product acetic acid and thereby force the reaction to near completeconversion of PE to N-acetyl-PE. However, complete conversion toN-acetyl-PE is not necessary. Both fluid and de-oiled lecithins areacetylated in solvents, especially hexanes or hexanes mixed with othersolvents selected from acetone, ethyl acetate or ethanol. Acetylation inthe presence of ethanol reduces any proclivity for acetylation of freehydroxyls of IPL and glycolipids. Under the conditions noted, PE toN-acetyl-PE conversion reaches 90%; no base catalyst is necessary, butthe usual inorganic, polymeric or small molecule bases may be employedprovided acetylation of OH groups is avoided. It is noted that mostsmall molecule base catalysts exemplified by NEt₃ should be excludedbecause of toxicity concerns.

Acetic anhydride, and other fatty acid anhydrides, surprisingly alsofacilitate extraction of PA. The mechanism for this facilitation is notunderstood but is ascribable to the potential in situ formation and highsolubility of the acyl-phosphoric mixed anhydride species in certainsolvents, notably EtOAc, alone and in mixture with other solvents; it isnoted that mechanistically no net chemical transformation of PA isexpected and none is overtly observed.

Solvents for Fractionation: Fractionation of lecithins by selectivesolvent extraction and precipitation was described above using hexanesto dissolve and short chain alcohols, particularly ethanol, to causeselective precipitation. Extraction with supercritical fluids isequivalent. Combinations of pairs of relatively non-polar and polarsolvents, are employed also, and include but are not limited tohydrocarbons, halogenated hydrocarbons, and low molecular weightalkylesters, ketones, and alcohols. Improved results are obtained whensuccessive extractions are carried out with different solvent pairs. Asan example, extraction and precipitation with the hexanes-ethanolfollowed by CHCl₃—CH₃OH, reduces the residual PC, PE, N-Acyl-PE, PA andother minor non-IPL materials in Total-IPL product compared with use ofhexane-ethanol alone. As expected from the Distribution Law, a singleextraction with a large solvent volume is less efficient than multipleextractions with fractional volumes. The relative volumes of eachcomponent of the solvent pair, in proportion to the lecithins, aredetermined by pilot experiments with a range of volumes of eachcomponent, and monitoring the efficiency and selectivity by TLC of thedissolved (supernatant) and precipitated (pellet) fractions. The saidrelative proportions are also determined by the ability of the pair toform two and multi-component azeotropes, density, initial cost and costof recovery and reuse. Extraction with EtOAc-EtOH is advantageous andpreferred. These advantages are related to differences in solventstrength and relatively solubility parameters. Extractions may becarried out in any suitable equipment, including Soxhlet types.

In place of selective solvent extraction and precipitation, functionallyequivalent operations are employed also; each aided by solvent andsolubility modifiers. The functionally equivalent operations include butare not limited to liquid-liquid, including supercritical fluid,partition. Preferably, these operations are carried out in conjunctionwith and aided by solvent and solubility modifiers. Solvent modificationis achieved by change of water-content, operational pH and ionicstrength, by inclusion of water, salt solution, acids or bases, forexample NaCl, acetic acid, phosphoric acid, and NEt₃. Use of fatty acidanhydrides, particularly acetic anhydride for solubility modification ofPE, is a critical operation as discussed in a previous section(N-Acetylation/Acylation).

Module 2. Selective Solvent Partition

Module 2 starts with Total-IPL from Module 1, and provides an IPLproduct that is substantially free of the polar glycolipids GSL;overall, the product, GSL-free IPL, is substantially free of GSL as wellas of PA, PC, PE, TG, FFA, SG, and SGE removed in Module 1. Module 1 andModule 2 may be integrated to provide the GSL-free product directly. Theresulting GSL-free IPL is more highly enriched in PI than Total-IPL; itcontains between 50-80% PI, and, may contain only a fraction of theoriginal PGL, PA, and Lyso-PI.

Typically, Total-IPL is dissolved in CHCl₃, CH₃OH and water to form twoliquid phases. The CHCl₃-rich phase is separated, and washed with freshCH₃OH-water phase. GSL partitions selectively into the upperaqueous-methanol layer, the washed CHCl₃-rich lower phase issubstantially free of GSL, and may be depleted in PGL as well. Ifinsufficient proportions of solvents to solute are employed, GSL and PGLalso separate out as a ppt. The layers are separated, the CHCl₃-richlower phase is filtered through Celite, evaporated under reducedpressure to obtain the GSL-free IPL product. The solvent partitioncomprising aqueous wash as outlined above is effective with a broadrange of polar and non-polar solvent pairs, in combination with water oran aqueous salt solution. The solvents are selected from but not limitedto hydrocarbon, halogenated hydrocarbon and alkylester, and ketone,short chain alcohol types, and are used in volume proportions that formtwo liquid phases with added water. Additionally, aqueous solutions ofalkali metal salts, including but not limited to sodium salts,preferably NaCl and Na₄-EDTA, are used to influence IPL partition andeffect concomitant cation-exchange to replace the endogenous Ca⁺⁺ andMg⁺⁺ counter-ions of IPL with Na⁺ cations. The cation content in theproducts, if desired, is determined by Atomic Absorption or equivalenttechniques.

Total-IPL solute to the less polar solvent w/v ratio ranges from 1:100to 1:5, preferably 1:0 to 1:20; as noted above, at low solvent ratios, aGSL/PGL-rich precipitate may form; when Module 1 and Module 2 areintegrated, the said w/v ratio up to 1:1000 is employed. Depending onthe solvent combinations selected, the GSL component selectivelydistributes into either the water-rich phase (e.g.,chloroform-methanol-water) or the water-poor phase (e.g.,hexane-ethanol-water). The GSL-free IPL product is isolated from theappropriate liquid phase, typically by evaporation under reducedpressure, or the product phase is used as such in Module 3.

Module 3. Selective Precipitation, Adsorption on Silica, Crystallization

Module 3 provides IPL products, which are partially or substantiallyfree of PGL in addition to being substantially free of GSL (removed inModule 2) as well as substantially free of PA, PC, PE, TG, FFA, SG, andSGE (removed in Module 1). PGL-free IPL contains 70-95% PI togetherwith, minor amounts of PGL, PA, Lyso-PI and other components. Pure PIfrom this module is 95-99% pure.

The module comprises selective precipitation for separation of IPLcomponents. Typically, the CHCl₃-rich phase from Module 2 containingGSL-free IPL, is cooled, diluted with cold CH₃OH and kept cold (0-5°C.). A precipitate rich in PGL is formed; the supernatant containing thePGL-free IPL is recovered by filtration (filter aid, Celite) orcentrifugation. This selective precipitation is repeated, if necessary.The filtrate or supernatant, is evaporated under reduced pressure toobtain the PGL-free IPL, or subjected to selective adsorption on silica.

Further purification to obtain pure PI is carried out by selectiveadsorption on silica of any remaining PGL and GSL from the PGL-free IPLproduct. Selective adsorption on silica is employed also to obtainPGL-free IPL product from Total-IPL or GSL-free IPL products. GSL-freeIPL in solution, preferably the filtrate or supernatant obtained at theselective precipitation stage, is treated with activated chromatographicgrade silica, the solution is filtered through a shallow bed ofchromatographic silica, and evaporated to obtain the PGL-free IPLproduct. Effective solvent mixture compositions are selected byexperiment. Mixtures of solvents, selected from those used in Module 2and exemplified by CH₃Cl and CH₃OH, with water or aqueous NH₄OH areeffective. The NaCl-washed CH₃Cl-rich phase or the supernatant fromselective precipitation at the last stage in Module 2 are preferred, andare used directly, or after some adjustment of solvent composition toallow selective adsorption on silica of components with greater polaritythan PI.

Adsorption on silica is carried out, and pure PI is obtained, by flashchromatography of Total-IPL or GSL-free IPL, or preferably of PGL-freeIPL, using CH₃Cl—CH₃OH or hexanes-IPA together with water or aqueousNH₄OH as the elution solvents. Pure PI is eluted, for example withCHCl₃—CH₃OH—NH₄OH (65:25:3 to 60:40:10, v/v/v).

Module 4. Characterization

The products are characterized by TLC. PI content is measuredgravimetrically using PI recovered by column chromatography on silica.The chromatographically isolated PI is characterized by MS, ¹H and ³¹PNMR, and, most critically by Optical Rotation (Specific and MolarRotation) data compared with pure Soy PI isolated from de-oiled soylecithin by chromatography, as discussed further under Module 6. Fattyacid composition of pure PI is obtained by acid catalyzed methanolysis,and isolation and identification by GC.

Module 5. Finishing

The products of Modules 1-3 preferably are isolated as sodium salts.Optionally, either PGL-free IPL or Pure PI in sodium salt form issubjected to crystallization under conditions that are known in theprior art.

Solvent residues are removed by continuous evacuation under high vacuum.Alternatively, and preferably the products are lyophilized from aqueousdispersions; these lyophilized products disperse easily in water forefficient intestinal absorption.

Module 6: Purification of IPL via Temporary Reversible ChemicalModification

The general process approach purifying IPL from natural lipids ispresented herein, and outlined in the Scheme in FIG. 6, with a focus onthe preparation of pure PI.

In Module 6, a natural lipid fraction that contains PI and issubstantially free of components with polarity lower than PI, is used asthe starting material. The starting material is treated (FIG. 6, Step a)with reagents to affect temporary O-substitution of the inositolhydroxyls of PI with base-stable acid-sensitive O-protecting groups. Thereaction yields an O-protected PI intermediate, which is O-deprotectedto regenerate PI (FIG. 6, Step c). The PI product is characterized toascertain unequivocally that it has retained the core stereo-structureof natural PI.

According to an aspect, the said PI-enriched natural lipid fraction isfrom seed phosphatides or commercial lecithins, particularly Soylecithin. The IPL compositions obtained in Modules 1, 2 or 3 of thepresent invention, are substantially free of components with polaritylower than PI, and are the most preferred starting materials.

The O-protected PI intermediates are designed to have a polarity lowerthan PI, and to be easily soluble in solvents that are poor solvents forPI and associated polar IPL components, and do show these properties.The intermediates are separated easily from the reaction mixture bysolvent extraction or flash chromatography on silica if components withsimilar polarity and solubility are not present. Lecithins that are notsubstantially free of components with polarity lower than PI, are lesssuitable starting lipid materials than IPL compositions similar to thoseprovided by Modules 1, 2 or 3.

The reagents for introducing the base-stable acid-sensitive O-protectinggroups are selected from mono-, di- and tri-functional reagents. Theseinclude but are not limited to acetal, ketal, and orthoformate types,exemplified by 3,4-dihydro-2H-pyran (DHP), cyclohexanone-dimethylketal,1-methoxycyclohexene, and trimethylorthoformate. The O-protecting groupsare exemplified by tetrahydropyranyl (THP), tetrahydrofuranyl (THF),4-methoxy-THP, cyclohexylidene, 2-methoxy-cyclohexyl, acetonide, andmethoxylmethyl (MOM). Most of these are introduced at relatively lowtemperatures in acid catalyzed reactions in anhydrous alcohol-freemedia. The MOM is introduced using MOMCl in the presence of a hinderedtert. amine. DHP, the reagent for introducing THP is available readily,and is inexpensive.

The O-protecting groups are subsequently removed (FIG. 6, Step c) bymild acid catalyzed hydrolysis or alcoholysis of the ketals withoutdamage to long-chain fattyacyl-esters and the overall PIstereostructure. Typically, the O-protected-PI is treated with aqueousalcoholic medium, in a particular aspect in aqueous ethanol ortert-butanol, acidified with p-TSA or equivalent insoluble acid, moreparticularly Nafion®. The acid catalyst is neutralized, according to anaspect, with aqueous NaHCO₃ solution. Undesirable side reactions areminimized by varying the solvent, acid, temp., and time parameters toobtain pure PI directly without need for extensive purification.Alternatively, anhydrous EtSH is used as the solvent and a Lewis acid,such as BF3-ether complex, or a protic acid, e.g. p-TSA, as the acidcatalyst at −20° C. to rt.

Very good to excellent yield are obtained in both the O-protection andO-deprotection steps.

Reaction conditions for O-protection and deprotection are employed whichdemonstrably yield O-protected PI intermediates and final PI productswith the natural 1D-1(1,2-diacyl-sn-glycero-3-phospho)-myo-inositolstereo-structure; these reaction conditions are defined and are aparticularly distinguishing feature of the present process.

It is noted that isopropylidene derivatives have been prepared from purePI obtained from Baker's yeast but these derivatives have not been usedas intermediates for recovering the intact original natural PI (Noda andKeenan, 1990)

p-TSA catalyzed treatment of pure Soy PI (FIG. 6) with a very large (100to 200×) molar excess of DHP, used both as solvent and reagent, yieldedone major product, identified as penta-O-THP-PI; the same product wasobtained in other experiments employing CH₂Cl₂ or other inert solventsand a similar large excess of DHP. In contrast, mixtures of partiallyO-protected O-THP-PI derivatives were formed when only a moderate excess(up to 10 molar) of DHP was employed.

The major product from reaction between pure Soy PI and an enormousexcess of DHP, was purified by chromatography on silicagel, usingchloroform-methanol-NH₄OH as eluent, and characterized by ES-MS, NMR andoptical rotation as the fully O-protected penta-O-THP-PI derivative(FIG. 6). NMR data indicated that the product was a mixture ofdiastereomers. It is noted that the reaction generates diastereomermixtures because a new asymmetric center is created with the formationof each of five O-THP links, and furthermore that the existence of thediastereomeric mixture does not affect the stereochemical integrity ofthe core PI stereo-structure in the product.

In the subsequent O-deprotection reaction step, significantly differentresults were obtained with the penta-O-THP-PI derivative obtained with alarge excess of DHP, contrasted with reaction of the aforementionedmixtures of partially O-protected O-THP-PI derivatives. CompleteO-deprotection (FIG. 6, Step c) of pure penta-O-THP-PI gave a productidentical with Soy PI. Notably, optical rotation data for the PIproduct, comprising the observed specific rotation [α]_(D)+6.2 to +6.3and molar rotation [φ]+52.8 to +53.9 calculated there from, wereidentical with data for a reference Soy PI, and comparable with theliterature values [α]_(D)+6.0 to +6.2 and molar rotation [φ]+51 to +52.8for Soy PI. Optical rotation data have been established as the cardinalstereo-chemically significant parameters for characterizing PI, and[α]_(D)+6.0 and molar rotation [φ]+51 have been established as benchmarks for the1D-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol absolutestereochemical structure (Aneja and Aneja, 2000; Aneja et al, 2002;Aneja, 2004, U.S. Pat. No. 6,737,536).

Complete O-deprotection of the aforementioned mixtures of partiallyO-protected O-THP-PI derivatives, obtained using only a moderate excessof DHP, gave a products which were similar to Soy PI but showed [α]_(D)and molar rotation [φ] significantly higher than normal values for SoyPI. The data indicate that these PI preparations are contaminated withthe 1L-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositoldiastereomer of natural PI for which [φ]+74 has been established as thebench mark value (Aneja and Aneja, 2000; Aneja et al, 2002; Aneja, 2004,U.S. Pat. No. 6,737,536).

The 1L-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositolcontaminant mentioned above is identical with1D-3-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol. Itsformation concomitant with the natural1D-1-(1,2-Di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol series isrationalized based on the relative reactivity of the various functionalgroups in PI in reaction with DHP and related reagents (Scheme, FIG. 7).Reactions of the four equatorial hydroxyls atinositol-3,4,5,6-positions, and the phosphoric acid OH, are expected tobe faster than axial 2-OH. With low to moderate molar proportions of theO-reactive reagents, partially O-protected intermediates are formed andlikely include cis-oriented 2-hydroxy-phosphotriesters carrying a THPesterified to the phosphoric residue; an example is provided in the3,4,5,6-tetra-O-THP-phosphotriester A shown in FIG. 7. The1,2-cis-configuration of the free 2-OH adjacent to the phosphotriesterat 1-O-position in A facilitates intra-molecular addition-eliminationvia B or equivalent intermediate structure and leads to isomerization tothe 2-phosphatidyl series structure C. The acid catalyzedteterahydropyranylations are reversible and lead to thecis-2,3-configured analogue of B, which in turn is able to equilibrateto the 3-phosphatidyl isomer (structure not shown). Because of themirror symmetry plane across C-2 and C-5 in myo-inositol derivatives,the 1D-3-phosphatidyl isomer is identical with 1L-1-phosphatidyldiastereomer. Formation and existence of the partially O-protectedintermediates with a free 2-OH is obviated when a very large excess ofthe reagents is employed under conditions which ensure rapid andcomplete O-protection.

It was mentioned earlier that optical rotation data have beenestablished as the cardinal stereo-chemically significant parameters forcharacterizing PI. A full discussion of the physicochemicalcharacterization and relationship to the absolute stereochemicalstructure of PI has been presented and the complete text of thepertinent publications, (Aneja and Aneja, 2000; Aneja et al, 2002;Aneja, 2004, U.S. Pat. No. 6,737,536) is incorporated herein byreference.

Important parameters pertinent to O-protection and O-deprotectioninclude the choice of reagent, reaction medium, catalyst, and conditionsfor introducing the base-stable acid-sensitive O-protecting groups. Inaddition, a relatively very large molar excess of the O-protectingreagent over the starting PI is essential for retaining the absolutestereo-structure, and is an integral and distinguishing feature of thepresent invention. A PI to O-protection reagent molar ratio between1:100 and 1:200, preferably 1:125 to 1:175 is employed.

In addition to the mono-functional reagent DHP discussed above (FIG. 6),the reaction of PI and the bi-functional reagent cyclohexanonedimethylketal catalyzed by p-TSA was utilized as well.2,3-Mono-O-cyclohexylidene-PI (not shown) together with2,3:4,5-di-O-cyclohexylidene-PI and 2,3:5,6-di-O-cyclohexylidene-PI areformed initially followed by the fully O-protected2,3:4,5-di-O-cyclohexylidene-6-(1-methoxycyclohexyl)-PI (not shown) and2,3:5,6-di-O-cyclohexylidene-4-(1-methoxycyclohexyl)-PI (not shown).Alternative fully O-protected O-cyclohexylidene-PI derivatives areprepared, for example by reaction of 2,3:4,5-di-O-cyclohexylidene-PIwith MOMCl in the presence of diethyl-isopropyl amine. CompleteO-deprotection of the various cyclohexylidene-PI derivatives gave PIidentical with Soy PI.

Treatment of Pure Soy PI in ether with methanolic tetrabutylammoniumhydroxide, gave 1D-1(sn-glycero-3-phospho)-myo-inositol (GPIns), andtreatment with phospholipase A₂ gave Soy Lyso-PI, useful as biochemicaland physiological precursors of PI; these two Soy PI derived productsare water soluble and efficiently absorbable in vivo.

Blends

The IPL products optionally are blended with antioxidants, and lipidswith synergistic bioactivity, including but not limited to the cardioatheroprotective mono- and diglycerides, ω-3 polyunsaturated andconjugated linolenic acid based lipids of algal and plant origin,phytosterols and derivatives.

Applications

The efficacy of various IPL products and compositions of the presentinvention is demonstrated by pertinent evaluation protocols describedpreviously, including but not limited to use as drug delivery vehicles(Lee et al, 1992), nutraceuticals and therapeutics for CNS disorders(Ferrari et al, 1993, U.S. Pat. No. 5,214,180) and CAD (Sparks, 2004;Burgess et al, 2005). GPIns and Lyso-PI are water soluble, providingefficient absorption and conversion in the chylomicrons into PI.

Applications of Soy PI in the IPL products and derived O-protected-PIsof this invention as starting materials and intermediates for synthesisof a wide variety of natural and synthetic IPL, and purification of SoyPI, are illustrated in FIGS. 2-6. The rationally designed PIderivatives, carrying functional groups at selected myo-inositolhydroxyl positions, as illustrated by the novel charge neutral, anionicand cationic structures in FIG. 2 are synthesized from2,3:4,5-di-O-cyclohexylidene-PI as a novel intermediate. The protocolsfor synthesis are similar to the novel synthesis of SoyPI-4,5-bisphosphate outlined in the scheme in FIG. 4, wherein the fullyO-protected 2,3:4,5-di-O-cyclohexylidene-6-O-MOM-PI is criticalintermediate. Fully O-protected PI derivatives, exemplified by2,3:4,5-di-O-cyclohexylidene-6-O-MOM-PI, constitute a novel classdesignated as “selectively deprotectable O-protected PI derivatives”which were used also for re-tailoring the fattyacyl residues in PI andderivatives by a deacylation cum reacylation cycle.2,3:4,5-Di-O-cyclohexylidene-PI is also a critical intermediate in thesynthesis of novel PI-based vehicles and drug-vehicle conjugates forspecific delivery to therapeutic targets in thephosphoinositide-dependent and allied metabolic and signaling cascades,illustrated in FIG. 3; it is also an intermediate for entry into thephosphatidyl-D-chiro-inositol series. Soy derived PI-4,5-bisphosphate isapplied, as a reservoir precursor of PI-3,4,5-trisphosphate and related3-phosphorylated phosphoinositides, in cell and nuclear membranepermeable derivative form, and as novel complexes with functionalpeptides and proteins forming reconstituted lipoproteins, illustrated bycomplexes with therapeutic proteins insulin, leptin and apoA.

The following examples are included to demonstrate certain illustrativeand preferred embodiments of the invention. It will be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent techniques discovered by the inventor to functionwell in the practice of the invention, and can thus be considered toconstitute certain of the preferred modes for practicing various aspectsof the invention.

EXAMPLES General Procedures

All operations and reactions were carried out under an inert gasblanket, usually N₂ or Argon, and with deoxygenated solvents. Solventswere removed and recovered by rotary evaporation under a vacuum at orbelow 35° C. bath temperature. Solvent-free materials were obtained bycontinuous evacuation under a high vacuum. The progress of separationsand reactions was monitored by TLC, using Silicagel G on glass or otherprecoated plates described in the examples. New synthetic compounds werecharacterized fully using products judged to be >99% pure by TLC.Satisfactory MS (ES, MALDI-TOF) and ¹H NMR (400 MHz) data conforming tothe assigned structure were obtained for all new compounds.

Fluid lecithin refers to commercial fluid soy lecithin (containing ca.10% PI, 37% TG), typically Alcolec S from American Lecithin Company.Further, de-oiled lecithin refers to commercial lecithin granules orpowder (containing ca. 14% PI, 3% TG), from American Lecithin Company,Central Soy, or Degussa.

Reference Soy PI was obtained from the alcohol insoluble fraction of soylecithin by chromatography on silica eluted with a gradient ofCHCl₃—CH₃OH—NH₄OH.

Example 1

This example shows that use of acetic anhydride facilitates extractionof PE and PA from lecithin into solvents that dissolve PC and othercomponents with lower polarity than PI, but do not dissolve IPL.

Comparative Solvent Extraction of Acetylated Lecithins and Lecithins

A mixture of de-oiled lecithin (100 mg) in EtOAc (2.5 ml) and aceticanhydride (10 μl) was held in a 70° C. water bath. After 60 min., EtOH(2.5 ml) was stirred in, and the mixture kept in 70° C. bath for 10min., removed from the bath, allowed to cool to rt, and centrifuged(5000 RPM, 20 min). A gummy pellet was obtained and was given the sametreatment as the starting lecithin, except that 4.5 ml EtOAc was used,and holding time at 70° C. water bath was 10 min. The second pellet(Total-IPL product, dry wt. 27.9 mg) contained, by TLC spot densities,PI (˜40%), PGL+GSL (˜50%), and small amounts of PA and components lesspolar than PI estimated at ca. 2% each.

Concurrent control experiments with de-oiled lecithin were conducted asabove except that in one experiment acetic anhydride was not used, andin second experiment acetic acid was used in place of the anhydride; thepellets after two precipitations in each (respective dry wts. 37.5, and37.1 mg) contained PI, PGL+GSL, and significant amounts of PA and PE.

In other experiments, the starting de-oiled lecithin was replaced byacetylated lecithin (180 mg, each) from Example 2; here also, PE and PAwere extracted out and Total-IPL product wt. and composition were verysimilar to those with de-oiled lecithins.

Example 2

This example illustrates the preparation of acetylated soy lecithin fromneat (solvent-free) fluid lecithin, and provides evidence that PI inacetylated lecithin remains unacetylated and retains the absolutestereochemical structure1D-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol of naturalPI.

Preparation of Acetylated Lecithin, and Characterization of Pure PIObtained

Fluid lecithin (100 g), in an evaporation flask (1 L, R.B.) was treatedwith acetic anhydride (7 ml), and left rotating on a rotary evaporatorunder a partial vacuum at 35-40° C. bath temp. The by-product aceticacid was distilled out during 2 hr. TLC showed that PE had been replacedalmost completely by a new spot with a higher Rf (no purple stain withNinhydrin spray reagent, blue stain with modified Zinzadze reagent),coinciding with authentic N-acetyl-PE (prepared from pure PE by reactionwith acetic anhydride with NEt₃ catalyst). Aliquots of this acetylatedlecithin were used in several experiments.

An aliquot of acetylated lecithin (ca. 2 g, in hexanes 2 ml) was stirredinto EtOH (160 ml) containing EtOAc (40 ml), the mixture left at 0-5° C.overnight. The supernatant was decanted off, the ppt dissolved inhexanes (6 ml), the solution stirred into cold EtOH (200 ml), held at0-5° C. for 1 hr, centrifuged (500 RPM, 20 min, 0-5° C.), and, thesupernatant decanted off. The pellet dissolved in CHCl₃-MeOH—H₂O (6 ml:1 ml: 0.6 ml), was applied to a chromatography column containing silica(50 g) packed in CHCl₃-MeOH—NH₄OH (80:20:2, v/v/v), eluted with the samesolvent, followed by CHCl₃-MeOH—NH₄OH (65:35:3, v/v/v) which eluted outPI. PI-containing fractions were identified by TLC and pooled,evaporated to dryness, and dried overnight under a continuous highvacuum, gave PI as NH₄-salt. Yield 232.9 mg. TLC, Silicagel G,CHCl₃-MeOH—NH₄OH (60:40:10, v/v/v), Zinzadze spray, and charring, singlespot R_(f)0.3. [α]_(D)+6.3 (c, 1.0, CHCl₃-MeOH 4:1); reference soy PINH₄-salt [α]_(D)+6.3 (c, 1.0, CHCl₃-MeOH 4:1); literature, soy PI[α]_(D)+6.0 (c, 0.83, CHCl₃; Colacicco and Rapport, 1967); literature,soy PI [α]_(D)+6.2 (c, 0.51, CHCl₃-MeOH 4:1; Aneja and Aneja, 2000).

NMR spectra (¹H and ³¹P) of pure PI from acetylated lecithin wereidentical with reference soy PI, and contained no CH ₃CO resonance peak.

Example 3

This example illustrates the preparation of the various IPL compositionsby each of consecutive Modules 1, 2 and 3, and the pertinent processparameters and conditions, starting with a fluid lecithin.

Preparation of IPL Products from Fluid Lecithin

To fluid lecithin (100 g) (commercial fluid soy lecithin containing ca.10% PI, 37% TG, from American Lecithin Company), diluted with acetone(30 ml), at rt, acetic anhydride (7 ml) was added with stirring. After75 min, TLC showed that most of the PE had been converted intoN-acetyl-PE. The reaction mixture was added slowly into stirred acetone(1 L). The supernatant was decanted off and the gummy ppt was rinsedwith acetone (100 ml). The rinsed ppt was dissolved in hexane (100 ml),the cloudy solution added to stirred EtOH (1 L), the supernatantdecanted off and the gummy ppt rinsed with EtOH (100 ml); thisprecipitation operation was repeated three more times using ethanol, andonce each with 1:9 acetone-EtOH and 1:1 acetone-EtOH. The ppt changedfrom gummy to finely powder when acetone was employed; it was filterablebut was isolated by centrifugation (5000 RPM, 20 min, 5° C.-15° C.). Thefinal pellet, Total-IPL, (23.7 g) contained, by TLC spot densities PI(40%), PGL+GSL (52%), PA (3-5%) and small amounts of Lyso-PI, PC, PE,N-acetyl-PE SGE, and related lipids, estimated at <1% each.

The final pellet, Total-IPL, was dissolved in CHCl₃ (200 ml), and mixedwith MeOH (200 ml) and 5% aqueous NaCl (120 ml). The lower CHCl₃ layerwas set aside. The upper aqueous-MeOH layer was washed with the lowerCHCl₃ layer from a solvent blank prepared by adding CHCl₃ (200 ml) toMeOH (200 ml) and 5% NaCl (120 ml). The CHCl₃ layer from this wash wasadded to the CHCl₃ layer set aside above. TLC of the upper layer(MeOH—NaCl phase) showed that the GSL was present as the predominantcomponent (a spot at the origin showing no blue stain with modifiedZinzadze phosphate visualization spray reagent, but charring darkly at110° C.). The lower layer (CHCl₃ phase) contained ca. 15.6 g material,the GSL-free IPL product; it contained PI (60%), PGL (30%), PA (5%) andsmaller amounts of Lyso-PI, PC, PE, N-acetyl-PE, SG, SGE and relatedlipids estimated at <2% each.

The combined CHCl₃ layer containing the GSL-free IPL product was mixedwith fresh CHCl₃ (133 ml), and then MeOH (1100 ml). The resulting pptwas filtered through Celite filter aid. The filtrate was diluted furtherwith CHCl₃ (500 ml) and treated with 5% NaCl (660 ml). The lower CHCl₃layer (ca. 1000 ml) was evaporated to obtain the PGL-free IPL product(7.2 g); it contained PI (˜95%), and small amounts of PGL, PA, Lyso-PI,PC, PE, N-acetyl-PE, SG, SGE and related lipids estimated at <1% each.

The ppt was recovered by rinsing the spent Celite with warm CHCl₃ (100ml), and evaporation (3.2 g); it contained PGL as the predominantcomponent (75%) with PI (23%) and PA (2%).

In a cognate experiment, Total-IPL product containing PI (38%), PGL+GSL(˜60%), and small amounts of PA and PC (<1% each) was partitioned inCHCl₃, MeOH, and 4% NaCl. To the lower CHCl₃ layer (18 ml) containing60-65% PI (36 mg) and 35-40% PGL+GSL, was added chromatographic gradesilica (2.5 g), in 0.5 g portions. TLC of the solution showed that thePGL+GSL spot density decreased successively with each addition ofsilica; the final composition of the supernatant was estimated as PI(90-95%) and PA (˜3%), with PGL (<1%).

In other related experiments, the CHCl₃-MeOH-aqueous phase partition wascarried out with Na₄ EDTA followed by aqueous NaCl.

Example 4

This example illustrates the preparation of Total-IPL product and thepertinent process parameters and conditions, starting with de-oiledlecithin.

Preparation of Total-IPL Product from De-Oiled Lecithin

De-oiled lecithin (100 g), was stirred into hot EtOAc (2.5 L) held at60-70° C., acetic anhydride (10 ml) was added with stirring. After 60min, EtOH (2.5 L) was added, mixed for 15 min, allowed to cool to rt andcentrifuged 4000 RPM, 20 min. The supernatant was decanted off, and thepellet suspended in EtOAc (4.5 L), treated with acetic anhydride (10ml), and after 30 min EtOH (2 L) was added and the mixture stored undernitrogen atmosphere over the weekend. The supernatant was separated bydecantation and centrifugation, and the pellet was suspended in EtOH,centrifuged, re-suspended in EtOAc and evaporated to dryness in rotaryevaporator under reduced pressure, followed by high vacuum. The product,Total-IPL, 17.5 g, contained, by TLC spot densities PI (˜65%), PGL+GSL(˜35%), and less than 2% total of other components.

In a cognate experiment, Total-IPL product was obtained containing PI(˜38%), PGL+GSL (˜60%), PA (˜1%), PC (˜1%) and minute amounts of othercomponents. Hexanes (2 ml), EtOH (1 ml), and de-ionized water (50 μl)were mixed with the aforementioned Total-PI product (17 mg) containingPI (38%). The resulting two liquid layers were separated. TLC showedthat the lower aqueous-EtOH layer contained mainly PI (˜80%) with GSL(˜7%), PA (˜5%), and small amounts of PC (˜2%) and SG (˜1%). The upperhexane layer contained mainly PGL+GSL (˜85%), PI (˜10%), with smallamounts of PA (˜2%) and PC (˜1%). About 67% (˜4 mg) of total PI was inthe aqueous-EtOH layer. Further, in comparative experiments, thequantity of added water was varied, and in others, different volumeratios of hexanes and EtOH were employed.

Example 5

This example illustrates the preparation of Total-IPL composition andthe pertinent process parameters and conditions, starting with de-oiledlecithin.

Preparation of Total-IPL Product from De-Oiled Lecithin

A solution of de-oiled lecithin (1 Kg) in hexanes (1 L), was dilutedwith EtOAc (2 L), the solution slowly added to stirred ethanol (7 L)containing water (350 ml), the mixture was centrifuged (3500 RPM, 20min, rt) and the pellet recovered. The pellet dissolved in hexanes (1.3L), and EtOAc (900 ml) was treated with acetic anhydride (40 ml),maintained at 40° C. for min, and the reaction mixture poured slowlyinto stirred ethanol (7 L). The operation comprising dissolution, andprecipitation was repeated three more times using 1.2 L hexanes and 700ml EtOAc, including twice with inclusion of acetic anhydride (40 ml),and once without it. The resulting pellet was suspended in EtOH (3.6 L),centrifuged, and dried under a vacuum. The final pellet (194.8 g),Total-IPL, contained PI (ca. 60-65%), PGL+GSL (ca. 35-40%), PA and otherminor components (<1% each) judged by TLC spot densities.

Flash chromatography of Total-IPL (8 g) on silica eluted withCHCl₃-MeOH—NH₄OH (65:3:3, v/v/v) gave PI (5.2 g; 98%).

Example 6 Soy PI-2,3,4,5,6-penta-O-tetrahydropyranyl ether (FIG. 6)

A mixture of pure Soy PI (5.96 g), DHP (100 ml) as reagent and solvent,and Molecular Sieves 4 Å (2.0 g) was cooled to 0-5° C., anhydrouspowdered p-TSA (0.2 g) added, the mixture allowed to warm to rt, andheld briefly at 40° C.; the progress towards the desired product wasmonitored by TLC to completion. An excess of NEt₃ was added followed byaqueous NaHCO₃, and the mixture evaporated under a vacuum to remove thevolatile components. The residue was extracted with CHCl₃, and theextract purified by chromatography on silicagel eluted with a gradientof CHCl₃—CH₃OH—NH₄OH gave Soy PI-2,3,4,5,6-penta-O-tetrahydropyranylether (7.36 g; yield 85%); −m/z 1253.6 (M−H), −m/z 1281.6, etc. (minormolecular species), [α]_(D)−11.49 (c 0.94, CHCl₃). A very minor producteluted subsequently, is tentatively considered to be eitherhexa-O-THP-Lyso-PI or Soy PI-3,4,5,6-tetra-O-tetrahydropyranyl ether.

Soy PI-2,3,4,5,6-penta-O-tetrahydropyranyl ether dissolved easily inalcohol, acetone, ethyl acetate or methanol.

In other experiments, Total-IPL product was used in place of pure PI.Further, O-protection reaction was performed in an anhydrous inertsolvent, for example CH₂Cl₂, CHCl₃, hexanes or EtOAc, using a similarlylarge molar excess of DHP. With a larger proportion of p-TSA, reactionwas rapid at 0-5° C. The reaction in neat reagent is highly exothermicwith a tendency to produce polymers if overheating occurs.

Example 7 Re-Conversion of Soy PI-2,3,4,5,6-penta-O-tetrahydropyranylether into PI (FIG. 6)

The title penta-O-THP-PI was treated with ethanol containing a trace ofp-TSA and the reaction monitored by TLC to completion. Purification bychromatography on silica gave a product identical with Soy PI, by ES-MS,NMR and [α]_(D) as in Example 2.

Characterization

The IPL products were characterized by TLC, MS, ¹H and ³¹P NMR, andoptical rotation (specific and molar), as appropriate. Comparativeoptical rotation data was used as the cardinal parameter for determiningabsolute stereo-structure and diastereomer composition. Fatty acidcomposition and distribution was determined by methanolysis, andphospholipase A₂ hydrolysis followed by methylation of free fatty acids,and identification and quantitation of fatty acid methyl esters by GC.

Finishing

As noted in the Examples, the products were finished by continuousevacuation under high vacuum. In selected cases, the products werelyophilized, generally as blends with antioxidant. The lyophilized IPLare easily dispersible in water.

Example 8

This example illustrates the preparation, characterization andapplications of IPL products comprising phosphatidyl-myo-inositolwherein one or more inositol hydroxyl group is phosphorylated, notablyof Soy 1-Phosphatidyl-myo-inositol-4,5-bisphosphate (7) (synthesisScheme 4) and analogues, as of key O-protectedphosphatidyl-myo-inositols as intermediates, and O-substitutedphosphatidyl-myo-inositol products.

Soy 1-Phosphatidyl-myo-inositol-4,5-bisphosphate (7)

Soy 1-Phosphatidyl-2,3:4,5-di-O-cyclohexylidene-myo-inositol (2). Thetotal IPL product containing about 65% soy phosphatidyl-myo-inositol(1), prepared as described in Example 4, was heated at ˜65° C. with alarge excess of cyclohexanone di(ethyl/methyl) ketal andp-toluenesulfonic acid (catalyst) under a slight vacuum. The reactionwas monitored by TLC and stopped after 3.6 h of heating by cooling,quenching with triethylamine, and saturated aq NaHCO₃ solution, andevaporated at 50° C. under reduced pressure. The residue was purified bychromatography on flash silica gel, eluted with CHCl₃/MeOH/NH₄OH (v/v/vratios 95:5:0.5, solvent 1; 90:10:1, solvent 2; 60:40:10, solvent 3. Thematerial in fractions eluted with solvent 2 contained the two isomericdi-O-cyclohexylidene derivatives (R_(f) 0.27 and 0.28; TLC, Silica gelG, chloroform/methanol/ammonia 87:11.5:1.5) was rechromatographed toobtain the pure 1-phosphatidyl-2,3:4,5-di-O-cyclohexylidene-myo-inositol(2); (TLC R_(f) 0.28); MS (m/z, negative mode) complex set near 993.7(M−1, acyl groups mainly palmitoyl and linoleoyl), complex set near1021.7 (M−1, acyl groups mainly stearoyl and linoleoyl). ¹H NMR (400MHz, CDCl₃) δ 0.88 (m, 6H, CH₃), 1.2-1.8 (m, 6H, cyclohexyl and fattyacyl CH₂), 1.96-2.1 (m, 4H, allylic CH₂), 2.24-2.34 (m, 4H, carbonylα-CH₂), 2.74-2.79 (m, 2H, diallylic CH₂), 3.364 (distorted t, J=9.7 and10.1 Hz, 1H), 3.698 (t, J=9.357 Hz, 1H), 3.85-3.94 (m, 1H), 3.99-4.12(m, 2H), 4.13-4.21 (dd, J=7.018 and 12.086 Hz, 1H), 4.22-4.28 (dd,J=5.068 and 8.577 Hz, 1H), 4.28-4.35 (m, 1H), 4.37-4.45 (dd, J=2.1 and11.5 Hz, 1H), 4.501 (t, J=4.679, 1H), 5.19-5.27 (m, 1H, glycerylmethine), 5.27-5.43 (m, 4H, olefinic H). ³¹P NMR (162 MHz, CDCl₃) δ−0.543. [α]_(D)=+15.4 (c 1.33, CHCl₃).

In cognate experiments, chromatographically purified soyphosphatidyl-myo-inositol (1) was used as the starting material, and the2,3-mono-O-cyclohexylidene and two isomeric di-O-cyclohexylidenederivatives of soy phosphatidyl-myo-inositol were characterized fully.The compound with structure1-phosphatidyl-2,3:4,5-di-O-cyclohexylidene-myo-inositol (2) (TLC R_(f)0.28) was distinguished from the1-phosphatidyl-2,3:5,6-di-O-cyclohexylidene-myo-inositol isomer bydirect comparison of the latter (TLC R_(f) 0.27) with authenticsynthetic1D-1-(1,2-di-O-oleoyl-sn-glycero-3-phospho)-2,3:5,6-di-O-cyclohexylidene-myo-inositol(prepared by phosphatidylation of2,3:5,6-di-O-cyclohexylidene-myo-inositol).

The reaction of (2) with succinic anhydride and4,4,-dimethylaminopyridine (DMAP), and complete deprotection of the2,3:4,5-di-O-cyclohexylidene groups gave the 6-)-Succinoyl

Soy1-Phosphatidyl-2,3:4,5-di-O-cyclohexylidene-6-O-methoxymethyl-myo-inositol(3). To a solution of (2) (4.1 g, 4.2 mmol) in dry CH₂Cl₂ (40 ml) anddiisopropyethylamine (˜26 ml) under N₂ atmosphere at 0° C. was addedchloromethyl methyl ether (MOMCl) (2.9 g, 36 mmol). After overnightreaction at room temperature (˜22° C.) the reaction solution was dilutedwith CHCl₃ (100 ml) and then washed with 1N HCl (50 ml), H₂O (2×50 ml)and aq sat NaHCO₃ (50 ml) where all solvents used were cold anddeoxygenated. The organic extract was dried over Na₂SO₄, evaporated at18° C. under reduced pressure then further dried overnight under highvacuum. The material was then purified by chromatography (silica gel,CHCl₃/MeOH/NH₄OH, 90:10:1, v/v/v) to obtain 3 (1.5 g, 35%; TLCR_(f)=0.30, (CHCl₃/MeOH/NH₄OH, 90:10:1, v/v/v). MS m/z, 1038.0. ¹H NMR(400 MHz, ˜2:1 CDCl₃/CD₃OD) δ 0.85-1.0 (m, 6H, CH₃), 1.2-1.8 (m, 62H,cyclohexyl and fatty acyl CH₂), 2.0-2.1 (m, 4H, allylic CH₂), 2.3-2.4(m, 4H, carbonyl α-CH₂), 2.7-2.8 (m, 2H, diallylic CH₂), 3.447 (s, 3H,methoxy CH₃), 3.47 (m, 1H, overlaps with latter peak), 3.98-4.14 (m,3H), 4.16-4.25 (m, 2H), 4.3-4.45 (4H, solvent OH overlaps this area),4.47-4.5 (m, 1H), 4.8-4.8 (m, 2H, MOM H), 5.2-5.3 (m, 1H, glycerylmethine), 5.3-5.4 (m, 4H, olefinic H). ³¹P NMR (162 MHz, ˜2:1CDCl₃/CD₃OD) δ −1.403. [α]_(D) data not obtained.

The fattyacyls in (3) are retailored by an alkali or lipase enzymecatalyzed hydrolysis and reesterification of the resulting free glycerylhydroxyls with any desired fattyacyls. Re-esterification with isostearicacid, a fully saturated fatty acid which is unlike stearic acid and isliquid at sub-zero temperatures, gives the 1,2-di-O-isostearoyl analogue(3A) of (3), which gives the series of products (4A)-(7A) analogous withcompounds (4)-(7).

Soy 1-Phosphatidyl-2,3-O-cyclohexylidene-6-methoxymethyl-myo-inositol(4). To a stirred solution of 3 (1.5149 g, 1.4575 mmol) in CHCl₃ (11.5ml) under N₂ atmosphere was added a solution of p-toluenesulfonic acid(443 mg, 2.33 mmol) in methanol (11.5 ml). After 33 min, when TLC showedcomplete disappearance of starting material. The reaction solution wascooled in an ice/water bath, quenched with triethylamine (477 mg), andthen aq NaHCO₃ (400 mg in 5 ml). The latter mixture was partitioned in aCHCl₃ (100 ml), MeOH (100 ml), and aq NaHCO₃ (1.3 g in 90 ml) mixture,the organic layer concentrated and purified by chromatography (silicagel, CHCl₃/MeOH/NH₄OH mixtures to obtain 4 (665 mg, 47.6, TLCR_(f)=0.34, CHCl₃/MeOH/NH₄OH, 75:25:2, v/v/v.) ¹H NMR (400 MHz, CDCl₃) δ0.85-1.0 (m, 6H, CH₃), 1.2-1.8 (m, 52H, cyclohexyl and fatty acyl CH₂),1.97-2.10 (m, 4H, allylic CH₂), 2.25-2.36 (m, 4H, carbonyl α-CH₂),2.70-2.84 (m, 2H, diallylic CH₂), 3.36-3.48 (m, 1H, overlaps with themethoxylmethyl), 3.432 (s, 3H, methoxy CH₃), 3.90-4.10 (m, 4H),4.11-4.23 (m, 2H), 4.25-4.32 (m, 1H), 4.32-4.43 (m, 2H), 4.68-4.78 (d,1H, J=6.7 Hz, MOM H), 4.79-4.88 (d, 1H, J=6.7 Hz, MOM H), 5.18-5.27 (m,1H, glyceryl methine), 5.27-5.43 (m, 4H, olefinic H). ³¹P NMR (162 MHz,CDCl₃) δ −2.168.

Soy 1-phosphatidyl-2,3-O-cyclohexylidene-myo-inositol and soy1-phosphatidyl-6-O-methoxymethyl-myo-inositol were obtained from otherlater fractions of the above chromatographic purification, and werecharacterized as novel compounds.

Methoxymethylation of (4) with diisopropyethylamine and MOMCl gave amixture from which the 4-O-MOM and the 5-O-MOM, and the 4,5-di-O-MOMderivatives of (4) were isolated, and respectively used for synthesis ofSoy 1-phosphatidyl-myo-inositol-5-phosphate, Soy1-phosphatidyl-myo-inositol-4-phosphate, and Soy1-phosphatidyl-myo-inositol-3-phosphate.

Reaction of (4), its 4-O-MOM or 5-O-MOM derivatives with diazomethanegives the corresponding phosphatidyl-OMe esters, and thesephosphotriester intermediates are O-phosphorylated and O-deprotected atthe myo-inositol-hydroxyls as described below for the phosphodiestercompounds (4) and (5); the phosphatidyl-OMe protection is lost and thephosphotriester reverts to phosphodiester on treatment withtriethylamine.

Soy1-Phosphatidyl-2,3-O-cyclohexylidene-6-O-methoxymethyl-myo-inositol-4,5-bis(9-fluorenylmethylphosphate)(5). To a mixture of 4 (256.1 mg, 0.2670 mmol) and 1H-tetrazole (527.5mg, 7.529 mmol) was added a solution of di-9-fluorenylN,N-diisopropyl-phosphoramidite (1.95 g, 3.74 mmol) in dry CH₂Cl₂ (8.8ml). The later mixture was left stirring and was judged complete by TLCafter 2 h. To the reaction solution at −20° is added a cold −20°solution of tetrabutylammonium periodate (1.50 g, 3.47 mmol) in CH₂Cl₂(3 ml). After 30 min the solution was warmed to and held at roomtemperature for 15 min, ethylene glycol (excess) was added, the mixturevortex mixed. The solution was partitioned in CHCl₃, MeOH, and 5% aqNaCl and the layers separated. The organic layer was evaporated at 0-5°C. and dried under vacuum and over P₂O₅. This dried material wasdissolved in dry CH₂Cl₂ (18 ml) and cooled to ca 0° C. beforetriethylamine (3.6 ml) was added and then warmed to rt (23° C.). Afterca 1.2 h at rt the reaction was judged complete by TLC, and was purifiedby column chromatography twice (silica gel, CHCl₃/MeOH/NH₄OH), andrepartitioned in CHCl₃, MeOH, and 1% aq NaCl, and the organic layerevaporated to yield 5 (87 mg, 27%; TLC R_(f)=0.14, CHCl₃/MeOH/NH₄OH,65:35:5 v/v/v). ¹H NMR (400 MHz, 3:2 CDCl₃/CD₃OD) δ 0.86-1.0 (m, 6H,CH₃), 1.047 (t, CH3 of n-butylammonium ion), 1.2-1.8 (m, 52H, cyclohexyland fatty acyl CH₂ which overlap with some n-butylammonium ion protons),1.98-2.11 (m, 4H, allylic CH₂), 2.2-2.3 (m, 4H, carbonyl α-CH₂), 2.7-2.8(m, 2H, diallylic CH₂), 3.15-3.23 (m, n-butylammonium N⁺CH₂), 3.323 (s,3H, methoxy CH₃), 3.95-4.8 (complex groups of m, 18H which overlap withsolvent OH), 5.20-5.28 (m, 1H, glyceryl methine), 5.28-5.43 (m, 4H,olefinic H), 7.22-7.30 (m, 4H, aromatic H), 7.31-7.39 (m, 4H, aromaticH), 7.67-7.78 (m, 8H, aromatic H). ³¹P NMR (162 MHz, 3:2 CDCl₃/CD₃OD) δ−0.396 (1P), 0.047 (2P).

Soy1-Phosphatidyl-2,3-O-cyclohexylidene-6-O-methoxymethyl-myo-inositol-4,5-bisphosphate(6). To a solution of soy1-phosphatidyl-2,3-O-cyclohexylidene-6-O-methoxymethyl-myo-inositol-4,5-bis(9-fluorenylmethylphosphate)(5) (75.5 mg, 0.0512 mmole) in dry CH₂Cl₂ (1 ml) was mixed triethylaminein an equal volume (1 ml) at rt (22° C.). After 5 days at roomtemperature almost all starting material was converted to 6 as judged byTLC. The reaction solution was purified by chromatography (silicic acid,200-325 mesh, acid washed-controlled particle size, 20:25:5:5CHCl₃/MeOH/NH₄OH/H₂O (v/v)) to obtain 6 (37.3 mg, 65.1%). TLCR_(f)=0.47, silica H treated with 1% potassium oxalate,CHCl₃/MeOH/NH₄OH+CO₂/H₂O, 20:25:5:5, v/v/v/v. [α]_(D)=+6.06 (c1.09,1:1:0.3 CHCl₃/CH₃OH/H₂O). ¹H NMR (400 MHz, 1:1:0.3 CDCl₃/CD₃OD/D₂O) δ0.84-1.02 (m, 6H, CH₃), 1.2-1.84 (m, 52H, cyclohexyl and fatty acylCH₂), 1.99-2.14 (m, 4H, allylic CH₂), 2.26-2.39 (m, 4H, carbonyl α-CH₂),2.74-2.86 (m, 2H, diallylic CH₂), 3.459 (s, 3H, methoxy CH₃), 4.0-4.12(m, 2H), 4.14-4.26 (m, 3H), 4.28-4.35 (m, 1H), 4.16-4.64 (m, 1H,overlapping with solvent OH), ca 4.5 (m, 3H, obscured by solvent OH),4.755 (br d, J=6.722 Hz, 1H, MOM group CH₂), 4.945 (d, J=6.741 Hz, 1H,MOM group CH₂), 5.24-5.44 (m, 5H, glyceryl methine and olefinic H), ³¹PNMR (162 MHz, 1:1:0.3 CDCl₃/CD₃OD/D₂O) δ −1.267 (1P), −0.747 (1P),−1.244 (1P).

Soy 1-Phosphatidyl-myo-inositol-4,5-bisphosphate (7). A solution of soy1-phosphatidyl-2,3-O-cyclohexylidene-6-O-methoxymethyl-myo-inositol-4,5-bisphosphate(6) (33.6 mg) in 1:1:0.3 CHCl₃/CH₃OH/HCl—H₂O (4 ml of deoxygenatedsolvent) was converted into the free acid form in CHCl₃, and evaporatedand dried under vacuum over P₂O₅. Ethanethiol (0.5 ml) was added to thedried material (26.7 mg) under N₂ blanket at rt (21.6° C.). After 2.6 hthe thiol was evaporated and methanol (2×0.5 ml) was added andevaporated under N₂ gas stream two times. After the residue was driedunder vacuum and purified by chromatography on silicic acid, 200-325mesh and partitioned in a Folch-type CHCl₃, MeOH, 1% aq NaCl mixture.The organic extract was evaporated under N₂ gas stream and dried undervacuum overnight to give 7 (13.3 mg, 44.5%). TLC R_(f)=0.32 (silica Htreated with 1% potassium oxalate, CHCl₃/MeOH/NH₄OH+CO₂/H₂O, 20:25:5:5v/v/v/v). MS (m/z, negative mode) 496.2 ((M−2)/2), 510.2 ((M+14−2)/2).¹H NMR (400 MHz, 1:1:0.3 CDCl₃/CD₃OD/D₂O) δ 0.8-1.05 (m, 6H, CH₃),1.2-1.4 (m, 38H, fatty acyl CH₂), 1.54-1.7 (m, 4H, carbonyl β-CH₂),2.0-2.15 (m, 4H, allylic CH₂), 2.25-2.4 (m, 4H, carbonyl α-CH₂),2.75-2.85 (m, 2H not 2H, diallylic CH₂), 3.64-3.7 (distorted dd, avg.J=2.77 & 9.76 Hz, 1H, inositol 3-H), 3.9-3.97 (m, 1H), 3.98-4.11 (m,4H), 4.17-4.24 (m, 2H), 4.27-4.36 (q, 1H, inositol 4-H), 4.4-4.47(distorted dd, avg. J=2.597 & 12.16 Hz, 1H, glycerol 1′-H, overlaps withsolvent OH), 5.2-5.45 (m, 5H, glyceryl methine & olefinic H). ³¹P NMR(162 MHz, 1:1:0.3 CDCl₃/CD₃OD/D₂O) δ 0.060 (1P), 1.622 (1P), 2.331 (1P).

The 1,2-di-O-isostearoyl analogue (7A) of (7), is prepared by synthesisfrom (3A) via (4A)-(6A) as described for synthesis of (7) from 3 via (4)to (6).

Both (7) and (7A) are designed and disclosed now as prototypicalphosphatidyl-myo-inositol-phosphates (PIP_(n)s) displaying lyotropicthermotropic mesomorphic transitions, at full hydration, matching thoseof PIP₂ from bovine brain; the latter which is the bench mark assaysubstrate for PI 3-kinase signaling enzyme family, is based onpolyunsaturated fattyacyls and is damaged quickly by non-specific aerialoxidation. The design of (7A) uses isostearic acid, a commercial mixtureof branched saturated 18-carbon fatty acids which remains liquid atsub-zero temperatures, and melts slowly (DTA/DSC) between −65 to −30.6°C. (cf. Arachidonic acid m.p. −49.5° C.). Longer and shorter chainanalogs of isostearic acid are obtained by standard chemistries. Thebroad group comprises diisostearoyl-PIP_(n)s and PIP_(n)s wherein atleast one of Alk¹CO and Alk²CO is isostearoyl and the other is theisostearic acid or a different saturated fattyacyl, or functionalizedfattyacyl, e.g., an ω-aminoalkanoyl, or N-substituted derivativethereof.

The group of PIP_(n) reagents disclosed herein, particularlydiisostearoyl-PIP₂ (7A) are designed specifically for matrix orsurface-spread micro- and nano-array kit-type applications as reagentsfor in vitro research studies, enzyme assays, and HTS systems based onphosphatidylinositol-phosphates as biological signaling messengers andtransducers.

All of the compositions and methods disclosed and claimed herein, can bemade and executed by those of ordinary skill in the art without undueexperimentation in light of the present disclosure. While thecompositions and methods of this invention have been described in termsof preferred embodiments, it will be apparent to those of skill in theart that variations may be applied to the compositions, methods and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope of the claimedinvention. More specifically, it will be apparent to those of ordinaryskill in the art that certain agents that are related chemically,structurally, functionally and/or physiologically, may be substitutedfor the particular agents described herein in order to yield the same,similar or otherwise beneficial results in accordance with theinvention. All such similar substitutes and modifications apparent tothose skilled in the art are to be included within the spirit, scope andconcept of the invention as defined herein and by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of isolating and purifying inositolphospholipids (IPL)comprising phosphatidylinositol (PI) from starting materials comprisingnatural plant source lipids or fractions thereof, wherein said naturalplant source lipids or fractions contain IPL, comprising the steps of:a. treating said starting materials with an anhydride and separating analcohol-insoluble precipitate material from an alcohol-soluble extractsolution; b. repeating said extraction and separation step (a) untilsaid precipitate is substantially free of components that are less polarthan PI, wherein said precipitate is a Total-IPL product; c. dissolvingsaid Total-IPL product in a two liquid phase aqueous wash, wherein saidliquid phases are separated by a solvent partition, wherein one of saidliquid phase comprises glycophytosphingolipids (GSL) and the other saidliquid phase is substantially free of GSL; d. filtering and evaporatingpart of said liquid phase that is substantially free of GSL to obtain asubstantially GSL-free IPL product; e. precipitating another part ofsaid liquid phase that is substantially free of GSL, wherein aprecipitate is formed comprising phytoglycolipids (PGL) and asupernatant is formed comprising a substantially PGL-free IPL product;f. recovering PGL-free IPL filtrate by filtration or PGL-free IPLsupernatant by centrifugation and evaporating said filtrate orsupernatant to obtain a substantially PGL-free IPL product; g. adsorbingon silica remaining PGL and GSL from said substantially PGL-free IPLproduct to obtain a substantially pure PI product; h. eluting saidsubstantially pure PI product with a solvent; i. purifying saidsubstantially PGL-free product by isolating in sodium salt form andsubjecting said sodium form product to crystallization; j. Optionally,step (c) by dissolving Total-IPL in CHCl₃, CH₃OH, and H₂O to form saidtwo liquid phase aqueous wash, wherein a CHCl₃-rich phase is separatedand washed with a fresh CH₃OH-water phase, and wherein said CHCl₃-richphase is substantially free of GSL; and wherein said substantiallyPGL-free and substantially GSL-free products comprise from about 1% w/wto about 5% w/w of the said PGL or GSL, respectively.
 2. The method ofclaim 1, wherein said natural source lipids are soybean lecithins.
 3. Asubstantially PGL-free IPL product prepared from soybean lecithinsaccording to claim
 2. 4. A method of preparing phosphatidylinositol (PI)products with natural 1D-1(1,2-diacyl-sn-glycero-3-phospho)-myo-inositolstereo-structure from a starting material, wherein said startingmaterial comprises said substantially GSL-free IPL product,substantially PGL-free IPL product, or substantially pure PI productobtained in claim 1, comprising the steps of: a. treating said startingmaterial with reagents to affect temporary O-substitution of theinositol hydroxyls of PI with base-stable acid sensitive O-protectinggroups to yield an O-protected PI intermediate; b. isolating andpurifying the said O-protected PI intermediate from the treatmentmixture by selective solvent extraction or by passing over/throughchromatographic silica; c. removing said O-protecting groups from saidO-protected PI intermediate by mild acid catalyzed hydrolysis oralcoholysis, and regenerating PI products with natural1D-1(1,2-diacyl-sn-glycero-3-phospho)-myo-inositol stereo-structure. 5.The method of claim 4 wherein the said base-stable acid-sensitiveO-protecting groups are selected from but are not limited to the groupcomprising tetrahydropyranyl (THP), tetrahydrofuranyl (THF),4-methoxy-THP, cyclohexylidene, 2-methoxy-cyclohexyl, acetonide, andmethoxylmethyl (MOM).
 6. An IPL compound, wherein the compound has theabsolute stereochemical structure1D-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol, and whereinone or both hydroxyl groups at myo-inositol positions 4- and 5-carryphosphoric acid residues or salts thereof, and wherein the fatty acidcomposition and distribution at the sn-glycero-1- andsn-glycero-2-positions are as in Soy phosphatidyl-myo-inositol with theshown 1-O-palmitoyl-2-O-linoleoyl motif as the predominant molecularspecies:


7. An IPL compound comprising the absolute stereochemical structure1D-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol, wherein oneor both hydroxy groups at myo-inositol positions 4- and 5-carryphosphoric acid residues or salts thereof, wherein the group at both thesn-glycero-1- and sn-glycero-2-positions is isostearoyl, and whereinsaid compound has the structure shown:


8. The IPL compound of claim 7 prepared by mono or bis-phosphorylationof a partially O-protected Soy phosphatidyl-myo-inositol.
 9. A partiallyO-protected Soy phosphatidyl-myo-inositol obtained by selectiveO-deprotection of O-protected Soy phosphatidyl-myo-inositol wherein theO-protecting groups are selected from orthogonally deprotectabletemporary protecting base-stable acid-sensitive groups.
 10. An IPLcompound, wherein the compound has the absolute stereochemical structure1D-1-(1,2-di-O-fattyacyl-sn-glycero-3-phospho)-myo-inositol, wherein the6-hydroxyl group carries at least one functional group or substituentwith negative, positive, or neutral net charge selected from the groupconsisting of phosphate, thiophosphate, sulphate, ω-aminoalkyl andcarboxylate, alkyl, and polyethyleneglycol, ω-carboxypropanoyl,(ω-amino-hexylamido)-ω-carboxypropanoyl-, and ω-carboxypropanoylpolyethyleneglycol ester groups, and their respective isoteric molecularand functional group analogues, and wherein said compound is structuredto be able to incorporate substituents at the 1,2-O-sn-glycero-positionsselected from the group consisting of AlkCO, Alk, and CH₃O(CH₂)_(n). 11.The IPL compound of claim 10, wherein the said IPL compound carriessubstituents at either one or both the 2-hydroxyl and the 3-hydroxylpositions, and wherein the said substituents are selected from a groupcomprising an alkyl or acyl, or replaced by F, Cl, CN, or N-acylamidogroups.
 12. The IPL compound of claim 10, wherein said respectiveisoteric molecular and functional group analogues of the phosphate inthe phosphatidyl residue comprise C-phosphonate and thiophosphonateresidues.
 13. The IPL compound of claim 7, wherein at least one of saidisostearoyl groups is replaced by a different saturated fattyacyl or afunctionalized fattyacyl selected from the group consisting ofω-aminoalkanoyl and N-substituted derivative thereof.
 14. A conjugateIPL delivery vehicle, comprising: a phosphatidylinositol derivative withthe following structure:

wherein R¹=Linoleoyl or isoStearoyl and R²=Palmitoyl or isoStearoyl; acontrolled stability and adjustable chain length linker attached at R³,wherein R³=

a drug that is bioactive towards therapeutic targets in thephosphoinositides cascade or downstream from thephosphoinositide-dependent metabolic and intracellular signalingpathways, and is attached at R⁴.
 15. The conjugate IPL delivery vehicleof claim 14, wherein the therapeutic target comprises protein kinases,phosphatases or allied signaling proteins.
 16. The method of claim 1,wherein said natural plant source lipids are seed phosphatides orlecithins, and the said fractions thereof are IPL-enriched.
 17. Themethod of claim 16, wherein said natural plant source lipids are soybeanlecithins.
 18. The method of claim 16, wherein said fractions thereofare PI-enriched.
 19. The conjugate IPL delivery vehicle of claim 14,wherein said phosphatidylinositol derivative comprises a therapeuticallyactive IPL delivery vehicle.
 20. The IPL compound of claim 10, whereinsaid compound is structured to be able to incorporate one or more of thefollowing modifying structural features: (a) the substituents at the1,2-O-sn-glycero-positions are selected from AlkCO, Alk, andCH₃O(CH₂)_(n), or (b) the substituent at the myo-inositol position 2-,or 3- is selected from the group consisting of Alkyl, Alkoxy, F, Cl, CN,CH2CH2OH, NHOCR.
 21. The method of claim 1, wherein said anhydride isselected from the group consisting of acetic, propionic and butyricanhydrides.